Process for determining 17-ketosteroids in urine and blood and solutions for carrying out this process



United States Patent 3,520,658 PROCESS FOR DETERMINING l7-KETOSTEROIDS IN URINE AND BLOOD AND SOLUTIONS FOR CARRYING OUT THIS PROCESS Nwaeze Anyanwu, 58-60 Rue de Carouge,

1200 Geneva, Switzerland No Drawing. Filed Jan. 27, 1967, Ser. No. 612,091

1 Int. Cl. G0lm 33/16 US. Cl. 23-230 Claims ABSTRACT OF THE DISCLOSURE Process for the colorimetric determination of 17-ketosteroids in urine or blood comprising hydrolysing urine or blood in a strongly acid media for 12 to 14 minutes with boiling, cooling to the ambient temperature and extracting with a slightly water-soluble solvent, washing this extract with an aqueous alkaline liquid, eliminating the solvent by evaporation, cooling, taking up the dry residue with a solution of m-dinitrobenzene in a solvent, adding a solution of potassium hydroxide, allowing the mixture to rest, diluting with a polar solvent, comparing the extinction of the resulting colored solution with a sample of defined concentration in order to know the initial concentration of 17-ketosteroids in the blood or the urine, the potassium hydroxide solution consisting of a stabilized solution in an anhydrous alcohol at a concentration of 3 to 6 N, the contact time between the mdinitrobenzene solution containing the residue and the potassium hydroxide solution before the dilution with the said polarsolvent being about 25 minutes, this polar diluting solvent being an anhydrous polar solvent.

It is already know to determine steroids having 19 carcon atoms and bearing a keto group in the 17 position, which are generally known under the name of neutral 17-ketosteroids, by making them react in a strong alkine medium with m-dinitrobenzent, which produces a violet color which remains stable for several hours. In the presence of an excess of m-dinitrobenzene, the optical density (O.D.) is proportional to the amount of 17-ketosteroids. Reference is hereby made to the following known conventional methods:

Borth, R.-Vit. & Horm. (1957) 259 Borth, R., Lunenfeld, B., and de Watteville, H.Fertil. &

Steril (1957) 233 Borth, R., et al.-Acta Endo 25 (1957) 33 Cahen, R. L., and Salter, W. T.J. Biol. Chem. 152

Callow, N. H., Callow, R. R., and Emmens, C. W.J.

Endocrinology 2 (1940) 88 Callow et al.J. Biochem. 32 (1938) 1312 Drekter, I. J., Heisler, A., Scism, G. R., Pearson, S., and McGawack, T. H. -J. Clin. Endo. Metab. 12 (1952) 55 Grandwohl, R. B. H.-Clin. Lab. Methods & Diagnosis 5th edit., vol. 2 (1956) 1323, Mosbyl Co., USA. Laboratory digest, vol. '(July 1956) No. 1; idem. vol. 20 (Aug. 1956) N0. 2; idem. vol. 20 (Oct. 1956) No. 4; idem. vol. 20 (Oct. 1956) No. 5

Hamburger, C.-In Emmens, C. W., ed. Hormone Assay chap. 7; Academic Press, New York, USA idem. Acta Endo. (Dec. 1958) Holtorff, A. F., and Koch, F. C.J. Biol. Chem. 135

Jayle, M. F., and Libert, O.Ann. Biol. Clin. 2 (1947) 93 Jayl, M. F.--Analiyses des Stroides Hormonaux vol.

1 et II (1962), Masson & Cie., Paris, France Kingsley, E. R., and Getchell, G.--Clin. Chem. 3 (1957) ice Klendshoj, N. C., et al.-I. Clin. Endo. & Metab. 13

Nathason, I. T., and Wilson, H.Endocrinology 33 (1943) Rodier, J., and Mallein, R.Manuel de Biochimie Pratique (1961) Librairie Maloine, Paris France Sundermann, J and Sundermann, 1., Jr.Lipids and the Steroid Hormones in Clinical Medicine (1960) 155 J.

B. Lippincott Co., USA

Zimmermann, W., Hoppe-SeylersZtschr. Physiol. Chem.

Zimmermann, W., Anton, H. U., Pontinus, D., Hoppe- Seylers-Ztschr. Physiol. Chem. 289 (1952) 1 However, these methods suffer from the following disadvantages:

(l) Colorimetric aqueous solutions are relatively unstable having a stability of at the most 7 minutes.

(2) The alcoholoic solutions usually used are not stable or the blank tests are not readable or must be made up each time on the day of use.

(3) Consequently, the work is laborious.

(4) They make impossible the practical realization of hormonal analysis at the same time as other chemical clinical tests.

(5) Most normal contents of steroids accumulated in man and determined by other technics are very generalized and render their clinical interpretation incoherent and diflicult and sometimes impossible.

(6) Some of these process have recourse to toxic reagents, for example, carbon tetrachloride and ethylene dichloride.

The present invention has for its object to obviate these disadvantages.

Owing to the use of stable solutions, it permits to make analysis in a reduced time of only about minutes. It makes possible to carry out other clinical chemical laboratory analysis at the same time as the hormone analysis while avoiding the use of all toxic reagents and solvents.

The invention has for its object a process for the colorimetric determination of 17-ketosteroids in urine or blood comprising hydrolysing urine or blood in a strongly acid media for 12 to 14 minutes with boiling, cooling to the ambient temperature and extracting with a slightly water-soluble solvent, washing this extract with an aqueous alkaline liquid, eliminating the solvent by evaporation, cooling, taking up the dry residue with a solution of m-dinitrobenzene in a solvent, adding a solution of potassium hydroxide, allowing the mixture to rest, diluting with a polar solvent, comparing the extinction of the resulting colored solution with a sample of defined concentration in order to know the inital concentration of 17-ketosteroids in the blood or the urine, the potassium, hydroxide solution consisting of a stabilized solution in an anhydrous alcohol at a concentration of 3 to 6 N, the contact time between the m-dinitrobenzene solution containing the residue and the potassium hydroxide solution before the dilution with the said polar solvent being about 25 minutes, this polar diluting solvent being an anhydrous polar solvent.

EXAMPLE Determination of 17-ketosteroids are made on 24 hourold urines from which an aliquot part is taken, generally from 2 to 10 ml. In a test tube equipped with a ground stopper, is placed 10 ml. of the urine to be analyzed and 2 ml. of concentrated hydrochloric acid or 2 ml. of 11 M sulfuric acid. This mixture is hydrolysed in a boiling steam bath for 13 minutes. It is cooled and then extracted by 20 ml. of ether. It is shaken about 50 times. The layers are 8, IN MG./24 HOURS enediamine-sulfate, ascorbic acid, benzoic acid and anhydrous sodium sulfate. The whole is well mixed in order to dissolve the dry extract in the test tubes. They are left in the absence of light for 25 minutes. There is added 5 ml. of anhydrous polar solvent, for example absolute alcohol. The whole is mixed. The optical density of these various mixtures is then read at 515 m (50 to 525 mg) on a spectrophotometer or a photometer. Test tube (UB) is read against absolute alcohol and U U and the standards against (RE). The concentration of the 17-ketosteroids in the urine, by comparing an average of the optical density of two urinary extracts (U and U less the absorption of (UB), is evaluated against a char-t indicating the average optical density as read on the spectrothe standard used as reference in the test. The optical density of a standard containing for instance 10 micro-grams of DHEA (by dehydroepiandrosterone is 0.095:0.01 as read on a Beckmann (DU model) spectrophotometer.

Obtained by the process of the invention, the normal contents in 17-ketosteroids in men and women established according to the age vary as shown in the two tables below.

ACCORDING TO AGE [Men] Limits Average ]Age Limits Average Age n R K P O Q H A Z108754 2211 O00fln-om&m S 4 20805420087055433322211111 m 54 44433333322222222 2222111 R m umQmamZZZZZZLLLLLLLLLLLLLLLLLL W U w A m A 4 g m 852076418764333009977777777 w 184070135420864110098888777 G m M .1 9 5 432100 8 7 0 m wme wm aa a 1a a waa aam M m .m .l 8 @4Ar33223222pk11l N 21 11.11111110000000000000ll L22121221LLLLLLLLLLLLLLLLLLL I S e g A 4 5 6 Lsh hohLlqmLipmlawohnhLlamLinmlflwohnh I 555555666666666677777777778 e g LLL Lrrunruo .J w 111 11111119999888777666555 A [Women] Limits ACCORDING TO AGE Limits Average I Age allowed to separate for 2 to 3 minutes and the aqueous layer is removed. The ethereal layer is then Washed successively with portions of 2 ml. of 4 N sodium hydroxide and 2 m1. of distilled water, rejecting the aqueous layers. There are taken then 3 aliquots, each of 4 ml. of the ethereal extract which are left in three test tubes marked UB,

U and U These three ethereal aliquots are evaporated to dryness by plunging them in a steam bath at temperature not much in excess of 40 C. The test tubes are wiped, cooled and colored at the same time as the aliquots of the samples of 10 to 40 microgrammes already evaporated to dryness at the moment of the hydrolysis of urine in the boiling water (B.M.). These test tubes are colored by adding in a test tube of urinary extract, 0.4 1, of absolute alcohol as a blank test for extract (UB) 15 photometer or photometer for various concentrations of and 0.4 ml. of an alcoholic solution of m-dinitrobenzene (1) in an empty and dry test tube used as a blank test for the reagents (RB) and the two test tubes of urinary extract as well as in all the test tubes of the aliquots of the standard, by adding in all the test tubes 0.3 ml. of the 3 20 to 6 N solution of potassium hydroxide in an anhydrous alcohol stabilized with a mixture of p-dimethylphenyl- NORMAL AMOUNTS OF URINARY 17-KETO STEROID Age NORMAL AMOUNTS OF URINARY 17KETO STEROID Age 0 0 In accord with this convention the preferred range of constituents for the stabilized alcoholic 3 N to 6 N KO=H is the following:

G./l. p-dimethylphenylenediamine sulfate .01 to 10 Ascorbic acid .01 to 100 Benzoic acid 0.01 to 200 Sodium sulfate 1 to 200 The alcohols used for this solution are preferably lower alkyl alcohols having 1 to 8 carbon atoms.

Among the advantages of the invention is the fact that the solution can be successively used in auto-dyalizing apparatus with colorimetric adaptations.

The process and solutions of this invention are also adapted to the determination of other steroids after transforming them into 17-ketosteroids. Such other steroids include a,fi-17-ketosteroids, total estrogens and estrogen fractions. The invention also is of value in determining the diagnostic relation of 17-ketosteroids and 17-hydroxysteroids of import in cancer detection.

What is claimed is:

1. A process for the colorimetric determination of 17- ketosteroids in urine or blood comprising hydrolyzing urine or blood by boiling in a strongly acid medium for 12 to 14 minutes, cooling the thus hydrolyzed solution to ambient temperature and extracting said solution with a slightly water-soluble solvent; washing this extract successively with a 4 N aqueous solution of sodium hydroxide and distilled water to eliminate undesirable chromogens and concentrating the purified extract to dryness by evaporating said solvent; cooling the dry residue and dissolving it in an alcoholic solution of m-dinitrobenzene and adding to the thus-colored solution a 3 to 6 N potassium hydroxide solution in an anhydrous alkanol having one to eight carbon atoms per molecule and stabilized with para-dimethyl-phenylenediamine sulfate, ascorbic acid, benzoic acid and sodium sulfate, allowing the mixture to rest and then diluting it with a polar solvent; and comparing With a spectrophotometer or with a photometer the extinction of the colored solution with the extinction of a Para-dimethylphenylenediamine sulfate 0.1 to 10 Ascorbic acid 0.01 to 100 Benzoic acid 0.01 to 200 Sodium sulfate 1 to 200 5. A reagent for performing the process for the colorimetric determination of 17-ketosteroids as claimed in claim 1, consisting essentially of a 3 to 6 N potassium hydroxide solution in anhydrous alkanol having one to eight carbon atoms per molecule and containing 0.01 to 10 g./l. of para-dimethylphenylenediamine sulfate, 0.01 to 100 g./l. of ascorbic acid, 0.01 to 200 g./l. of benzoic acid and 1 to 200 g./l. of sodium sulfate.

References Cited Cahen, R. L. et al. Journal of Biochemical Chemistry, vol. 152, pp. 489-99 (1944).

Chem. Abstracts, Subject Index, vol. pp. 256 8.5- 25695 (1966). Merck Index, 7th Edition, pp. 106, 133, 962 (1960).

Wilson, H. et al., Endochrinology, vol. 41, pp. 41721 (1947).

MORRIS O. WOLK, Primary Examiner E. A. KATZ, Assistant Examiner US. Cl. X.R. 252408 

